Cell stimulation, staining, and visualization are common techniques in both clinical and research settings. Cell stimulation and staining methods frequently involve the use of antibodies or other reagents that are expensive or available in limited quantities. Immunofluorescence staining is typically performed on cells adhered to glass slides, resulting in the need for a large volume of reagents. Moreover, cell stimulation and staining are frequently performed in parallel under a number of different conditions (e.g., time course, active agent, or antibody used). Such complex methods also make such cell assays labor intensive and low throughput.
Fluorescence activated cell sorting (FACS) and enzyme linked immunosorbent assay (ELISA) allow for screening of a large number of cells under a variety of conditions, frequently using smaller volumes or quantities of reagents than are required for immunofluorescence staining of cells on tissue culture slides. However, cells can only be sorted and observed as a group using both FACS and ELISA. Single cells cannot be tracked over time, as is possible using microscopy.
In order to track single cells over time in response to various conditions, a microfluidic device for fast and parallel single-cell based assays has been developed (Yun and Yoon 2005. Biomed. Microdev. 7:35-40). The device is designed to passively capture single cells or beads on multiple cell positioning sites by a pre-defined fluidic stream. The apparatus allows for the injection of specific reagents into each isolated single cell. Each cell is “captured” and held in place covering a hook shaped drain channel that allows the capture of single cells. Drugs or other agents can be injected through the drain channel and responses of single cells can be watched over time. It is not known what stress pathways might be activated by the positioning of the cell in the top of the drain channel or how long a cell would be viable under such conditions.
An elastomeric device has been developed for studying cell fate that allows for cell culture in an array of individual microwells (Chin et al., 2004. Biotechnol. and Bioeng. 88:399-415). Cells are located within individual wells that are all exposed to the same media. Therefore, although individual cells can be tracked over time, they are all exposed to the same stimuli.
Elastomeric devices have been developed for use in biological studies (for review see Sia and Whitesides 2003. Electrophoresis 24:3563-3576). Such devices are can be biocompatible and can be prepared relatively easily and inexpensively by methods well known to those skilled in the art (See e.g., reviews Whitesides et al., 2001. Ann. Rev. Biomed. Eng. 3:335-373, incorporated herein by reference).
Methods for fabrication of higher order structures to control fluid flow have also been developed. Unger et al. (2000. Science 288:113-116, incorporated herein by reference) demonstrated that elastomeric layers could be readily assembled to create channels regulated by elastomeric valves. Using such a layered design, miniaturized, elastomeric, computer-controlled microfluidics devices have been developed. Thorsten et al. (2002, Science 298:580-584, incorporated herein by reference) teach a high-density microfluidic silicone chip containing plumbing networks with thousands of binary, micromechanical valves, and hundreds of individually addressable chambers. Although fluids can be loaded and mixed using the device, it is not large enough to accommodate eukaryotic cells and seeding of cells with an even distribution would not be possible using the device. Moreover, precise control of fluid flow through channels is somewhat limited.
Gu et al. (2004, PNAS 101:15861-15866) teach a computerized microfluidic cell culture apparatus using elastomeric channels and Braille displays to control flow of fluids from reservoirs for patterning or mixing. This simplifies the fabrication of the device, but substantially limits the number of channels that can be accessed through single ports, and limits the density of the valves in the device, both of which decrease the throughput of the device.
There is a need for a device and method to allow for high throughput screening of living cells using a minimal quantity of reagents wherein the fate of individual cells can be followed over time.